1. Technical Field
The invention relates to administering polyamide nucleic acid oligomers to living cells such that the polyamide nucleic acid oligomers engender a sequence specific biological response.
2. Background Information
Polyamide nucleic acids (PNAs) are DNA analogs containing neutral amide backbone linkages. Unlike DNA oligomers, PNA oligomers can bind DNA by displacing one strand of the duplex to form a stable D-loop structure (Peffer et al., Proc. Natl. Acad. Sci. USA 90:10648-10652 (1993) and Mxc3x8llegaard et al., Proc. Natl. Acad. Sci. USA 91:3892-3895 (1994)). Interestingly, binding of PNA oligomers to DNA is independent of DNA strand polarity, allowing PNA oligomers to bind in both parallel and anti-parallel fashion (Egholm et al., Nature 365:566-568 (1993) and Peffer et al., Proc. Natl. Acad. Sci. USA 90:10648-10652 (1993)). In addition, PNA oligomers are less susceptible to enzymatic degradation (Demidov et al., Biochem. Pharmacol. 48:1310-1313 (1994)) and bind RNA with higher affinity than analogous DNA oligomers. Taken together, these properties suggest that PNA oligomers have great potential in both antigene and antisense approaches for regulating gene expression.
The success of an oligonucleotide analog as an antigene or antisense agent requires that the oligonucleotide be taken up by cells in reasonable quantities such that the oligonucleotide reaches its target at a sufficient concentration. PNA oligomers, however, have low phospholipid membrane permeability (Wittung et al., FEBS Letters 365:27-29 (1995)) and have been reported to be taken up by cells very poorly (Hanvey et al., Science 258:1481-1485 (1992); Nielsen et al., Bioconjugate Chem. 5:3-7 (1994); Bonham et al., Nucleic Acids Res. 23:1197-1203 (1995); Gray et al., Biochem. Pharmacol. 48:1465-1476 (1997)), which would appear to limit their potential uses in antigene and antisense approaches.
Recent strategies devised to improve cellular uptake of PNA oligomers involve conjugating other molecules to PNA sequences. Specifically, conjugating a small peptide sequence that binds the insulin-like growth factor 1 receptor (IGF1R) to a PNA oligomer increases cellular uptake of labeled PNA sequences by IGF1R-expressing cells, whereas conditions using unconjugated PNA sequences or cells lacking IGF1R show negligible cellular uptake (Basu S. and Wickstrom E., Bioconjugate Chem. 8:481-488 (1997)). These results suggest that conjugating receptor ligand molecules to PNA oligomers can increase cellular uptake; however, the ability of these receptor ligand-conjugated PNA oligomers to influence biological activity once inside the target cells remains unknown. Further, PNA oligomers will gain entrance only into cells expressing that particular targeted receptor. Thus, an appropriate ligand molecule would have to be designed and coupled to PNA oligomers for each cell type of interest. In addition, the level of receptor expression can influence the permeability of ligand-conjugated PNA oligomers.
The use of PNA oligomers to manipulate brain protein expression, an approach that would greatly aid the understanding of brain function as well as neurological disease, has an additional problem. The endothelial wall of capillaries in both brain and spinal cord creates a barrier (blood-brain barrier; BBB) that excludes the uptake of molecules into these organs. Although specialized transport systems operate within the BBB to allow certain circulating molecules to cross, many pharmaceutical agents are not recognized and thus have poor BBB permeability. This appears true for PNA molecules since the transport of PNAs across the BBB is reported to be negligible (Pardridge et al., Proc. Natl. Acad. Sci. USA 92:5592-5596 (1995)). Therefore, PNA oligomers targeting brain proteins administered outside the central nervous system need to cross two barriers, the BBB and the plasma membrane of individual cells within brain, whereas PNA oligomers administered directly into brain need to cross one barrier, the plasma membrane of individual cells.
Various drug delivery strategies can circumvent the BBB permeability problem (Pardridge, Pharmacol. Toxicol. 71:3-10 (1992); Pardridge, Trends Biotechnol. 12:239-245 (1994)). For example, PNA molecules can undergo transport through the BBB when the amino terminus is biotinylated and linked to a streptavidin conjugated monoclonal antibody specific for transferrin receptor (OX26-SA; Pardridge et al., Proc. Natl. Acad. Sci. USA 92:5592-5596 (1995)). The OX26-SA antibody delivers linked molecules to the brain presumably by receptor-mediated endocytosis, given the high transferrin receptor concentrations located on the BBB. These studies suggest that antibody-conjugation strategies provide a mechanism for PNA oligomers to cross the BBB. No data, however, exist as to whether the biotinylated PNA linked to OX26-SA actually enters cells or not. In addition, the utility of PNA delivery methods that rely on conjugating other molecules to PNA oligomers remains unclear since these other molecules may influence the desired functionality of particular PNAs.
The present invention relates to PNA oligomers that influence biological activity in a sequence specific manner. Specifically, this invention relates to the discovery that PNA oligomers administered extracellularly cross biological barriers and elicit a sequence specific biological response in living cells. This discovery is in direct opposition to the current understanding of the physical properties of PNA oligomers and has far-reaching implications for both gene therapy and research purposes. Further, extracellularly administering PNA oligomers to living cells circumvents the need to micro-inject PNA oligomers directly into cells as well as the need to permeabilize cells. In addition, this invention provides for the treatment of cells in vivo such that a behavioral response is observed in an organism. Thus, this invention describes methods and materials that allow any polypeptide to be manipulated and studied in living cells. For example, the expression of a specific polypeptide can be knocked-out in adult organisms for the duration of PNA oligomer treatment. In addition to greatly aiding the advancement of basic scientific research, this ability to manipulate polypeptide expression and thus function in a sequence specific manner is clearly beneficial to gene therapy approaches involving the treatment of cancer, aging, behavioral diseases, infections, and auto-immune diseases.
In addition, this invention relates to the following two discoveries: 1) antisense PNA oligomers administered to an animal cross biological barriers and modulate translation in a sequence specific manner, and 2) sense PNA oligomers administered to an animal cross biological barriers and modulate transcription in a sequence specific manner. Thus, this invention provides two powerful in vivo methods for manipulating polypeptide expression in a sequence specific manner. Clearly, having the ability to modulate two distinct steps involved in polypeptide synthesis will further the advancement of basic scientific research and provide useful methods for treating disease. The term xe2x80x9cantisense PNA oligomerxe2x80x9d refers to any PNA oligomer having sequence specificity for an RNA molecule found within a cell. The term xe2x80x9csense PNA oligomerxe2x80x9d refers to any PNA oligomer having sequence specificity for a region of nucleic acid that can be used as the template strand during transcription, including reverse transcription.
This invention also relates to the discovery that a mismatch PNA oligomer crosses biological barriers and engenders a biological response to a degree less than that engendered by a PNA oligomer targeting the same sequence but having, for example, no base pair mismatches. In other words, this invention relates to the discovery that the degree to which a biological response is engendered can be modulated by changing the sequence of a particular PNA oligomer to introduce base pair mismatches with a target sequence. Having the ability to alter the degree of an engendered biological response provides a useful mechanism for obtaining a particular desired effect. The term xe2x80x9cmismatch PNA oligomerxe2x80x9d refers to any PNA oligomer having a sequence that contains at least one base pair mismatch with respect to a target sequence. The term xe2x80x9ctarget sequencexe2x80x9d refers to any purine/pyrimidine sequence to which another purine/pyrimidine sequence (e.g., PNA, RNA, or DNA oligomer) exhibits sequence specificity.
Further, this invention relates to the discovery that PNA oligomers can be detected in a biological sample collected from an animal. Specifically, the presence and amount of a PNA oligomer can be determined by adding a sequence specific probe to a biological sample and examining that sample for the presence of probe/PNA oligomer complex. The probe/oligomer complex provides an indication of the presence of the PNA oligomer. Detecting PNA oligomers in a biological sample such as blood provides a useful method for monitoring the presence and concentration of a particular PNA oligomer during a treatment regimen. For example, quantifying the concentration of a particular PNA oligomer in a patient""s blood or tissue can allow clinicians to adjust the amount of PNA oligomer administered to the patient such that the desired concentration and biological response is achieved or maintained.
In general, the invention features methods of treating an animal containing a cell. The methods include administering a PNA oligomer to the animal under conditions such that the PNA oligomer enters the cell (e.g., a nervous system cell and a gastrointestinal cell) and modulates transcription in a sequence specific manner. The administration can be an extracranial (e.g., intraperitoneal, intravenous, and oral) or intracranial administration. The PNA oligomer can be carrier-free and capable of crossing the blood-brain barrier of the animal. The PNA oligomer typically has sequence specificity for at least a portion of a coding strand of DNA within the cell. That portion of a coding strand of DNA can regulate, or be a template for, synthesis of an RNA molecule (e.g., mRNA). Specific PNA oligomers can include oligomers having sequences such as set out in SEQ ID NO:s 13 and 14. The modulation of transcription can reduce expression of a polypeptide within the animal and/or cause a physiological change in the animal. The polypeptides can be expressed intracranially or extracranially (e.g., in the gastrointestinal tract) in the animal. In addition, the polypeptides expressed in the animal can include receptors (e.g., neurotensin receptors). Further, the methods can involve administering a second PNA oligomer to the animal under conditions such that the second PNA oligomer enters at least one cell within the animal and modulates translation in a sequence specific manner.
Another aspect of the invention features an article of manufacture that combines packaging material and a PNA oligomer. The packaging material includes a label or package insert indicating that the PNA oligomer can be administered to an animal to modulate transcription in a sequence specific manner.
In another aspect, the invention features methods of treating an animal containing a cell. The methods include administering a PNA oligomer to the animal under conditions such that the PNA oligomer enters the cell (e.g., a nervous system cell) and engenders a biological response in a sequence specific manner. In this case, the PNA oligomer has sequence specificity for a target sequence within the cell while possessing at least one base pair mismatch with that target sequence. A specific PNA oligomer can include a PNA oligomer having the sequence set out in SEQ ID NO: 12. The target sequence can be at least a portion of a coding strand of DNA within the cell. That portion of a coding strand of DNA can regulate, or be a template for, synthesis of an RNA molecule that, for example, regulates expression of or encodes a polypeptide. The biological response can be characterized by a physiological change in the animal.
The invention also features methods for identifying a mismatch PNA oligomer that engenders a modulated biological response in a subject animal having a target sequence. A modulated biological response is in comparison to a biological response engendered by a reference PNA oligomer. The methods include obtaining reference information about the biological response engendered by the reference PNA oligomer administered to a reference animal having the target sequence. This reference PNA oligomer has sequence specificity for the target sequence. The methods also include determining test information about the biological response engendered by a test PNA oligomer administered to the subject animal. The reference and test PNA oligomers have different sequences and the test PNA oligomer has sequence specificity for the target sequence while possessing at least one base pair mismatch with that target sequence. In addition, the methods include identifying the test PNA oligomer as a mismatch PNA if the test PNA engenders the modulated response.
Another aspect of the invention features kits for treating an animal containing a cell with a target sequence. The kits contain a plurality of PNA oligomers with each PNA oligomer having sequence specificity for the target sequence while possessing a sequence different from the sequence of the other PNA oligomers. One of the PNA oligomers can have a sequence completely complementary with the target sequence. At least one of the PNA oligomers can have a sequence having at least one base pair mismatch with the target sequence. The plurality of PNA oligomers can be a series of PNA oligomers having incrementally increasing degrees of base pair mismatch with the target sequence.
In another aspect, the invention features methods for treating an animal containing a cell with a target sequence. The methods include selecting a first PNA oligomer from a kit. The kit contains a plurality of PNA oligomers with each PNA oligomer having sequence specificity for the target sequence while possessing a sequence different from the sequence of the other PNA oligomers. The methods also include administering the first selected PNA oligomer to the animal under conditions such that the first selected PNA oligomer enters the cell and engenders a biological response in a sequence specific manner. The methods can further include selecting a second PNA oligomer from the kit and administering that second selected PNA oligomer to the animal under conditions such that the second selected PNA oligomer enters a cell in the animal and engenders the biological response to a modulated degree compared to the response engendered by the first selected PNA oligomer.
Another aspect of the invention features an article of manufacture that combines packaging material and a PNA oligomer. The packaging material includes a label or package insert indicating that the PNA oligomer can be administered to an animal under conditions such that the PNA oligomer engenders a biological response in a sequence specific manner. The PNA oligomer can have sequence specificity for a target sequence while possessing at least one base pair mismatch with the target sequence.
Another aspect of the invention features methods for detecting a PNA oligomer in a biological sample collected from an animal. The methods include providing an oligonucleotide to the sample. The oligonucleotide has sequence specificity for the PNA oligomer. The methods also include examining the sample for the presence or absence of an oligonucleotide/PNA oligomer complex. The oligonucleotide/oligomer complex provides an indication of the presence of the PNA oligomer. The PNA can be carrier-free. The biological sample can be a tissue sample (e.g., brain, liver, kidney, and heart tissue) or a blood sample. The oligonucleotide can be a labeled oligonucleotide (e.g., radioactively labeled). The examining step can include separating the oligonucleotide from any of the oligonucleotide/PNA oligomer complexes. The separation can be an electrophoretic separation.
Another aspect of the invention features an article of manufacture that combines packaging material and an oligonucleotide. The packaging material includes a label or package insert indicating that the oligonucleotide can detect a PNA oligomer in a biological sample collected from an animal. The oligonucleotide has sequence specificity for the oligomer.
Another aspect of the invention features methods of treating animals by orally administering PNA oligomers under conditions such that the PNA oligomers engender a biological response in a sequence specific manner. The PNA oligomers can be carrier-free and are capable of crossing biological barriers such as the plasma membrane of cells and the blood-brain barrier of an animal. The PNA oligomers typically have sequence specificity for a nucleic acid sequence that encodes a polypeptide or regulates the expression of a polypeptide expressed in the animal. The polypeptides can be expressed intracranially or extracranially. Polypeptides expressed in the animal can include transporters as well as those polypeptides that participate in cell signaling. Cell signaling polypeptides include polypeptides that participate in opioid signaling. Opioid signaling polypeptides include opioid receptors, for example morphine and neurotensin receptors. Transporter polypeptides can be serotonin transporters. Extracranially expressed polypeptides include polypeptides expressed outside the brain and cranium, for example in the gastrointestinal tract. Specific PNA oligomers can include oligomers having sequences such as set out in SEQ ID NO:s 1, 2, and 3. The biological response engendered by the extracellular administration of PNA oligomers can be a modification, for example a reduction, of polypeptide expression. Biological responses also can be characterized by a physiological change in an animal.
Another aspect of the invention features an article of manufacture that combines packaging material and PNA oligomers. The packaging material includes a label or package insert indicating that the PNA oligomers can be orally administered to an animal for the purpose of engendering a biological response in a sequence specific manner.
Another aspect of the invention features methods of treating living cells by extracellularly administering PNA oligomers under conditions such that the PNA oligomers engender a biological response in a sequence specific manner. The PNA oligomers can be carrier-free and can be administered in vivo to an animal. The PNA oligomers are capable of crossing biological barriers such as the plasma membrane of cells and the blood-brain barrier of a living organism. The PNA oligomers typically have sequence specificity for a nucleic acid sequence that encodes a polypeptide or regulates the expression of a polypeptide. The polypeptides can be expressed intracranially or extracranially in a living organism. Intracranially expressed polypeptides can include transporters as well as those polypeptides that participate in cell signaling. Cell signaling polypeptides include polypeptides that participate in opioid signaling. Opioid signaling polypeptides include opioid receptors, for example morphine and neurotensin receptors. Transporter polypeptides can be serotonin transporters. Extracranially expressed polypeptides include polypeptides expressed outside the brain and cranium, for example in the gastrointestinal tract. Specific PNA oligomers can include oligomers having sequences such as set out in SEQ ID NO:s 1, 2, and 3. The biological response engendered by the extracellular administration of PNA oligomers can be a modification, for example a reduction, of polypeptide expression. Biological responses also can be characterized by a physiological change in an animal.
Another aspect of the invention features a method of screening PNA oligomers for the ability to engender a sequence specific biological response by extracellularly administering PNA oligomers to living cells. The PNA oligomers can be carrier-free and can be administered in vivo to an animal. The PNA oligomers are capable of crossing biological barriers such as the plasma membrane of cells and the blood-brain barrier of a living organism. The PNA oligomers typically have sequence specificity for a nucleic acid sequence that encodes a polypeptide or regulates the expression of a polypeptide. The polypeptides can be expressed intracranially or extracranially in a living organism. Intracranially expressed polypeptides can include those polypeptides that participate in cell signaling. Cell signaling polypeptides include polypeptides that participate in opioid signaling. Opioid signaling polypeptides include opioid receptors, for example morphine and neurotensin receptors. Extracranially expressed polypeptides include polypeptides expressed outside the brain and cranium, for example in the gastrointestinal tract. The biological response engendered by the extracellular administration of PNA oligomers can be a modification, for example a reduction, of polypeptide expression. Biological responses also can be characterized by a physiological change in an animal.
Another aspect of the invention features a method of identifying polypeptide function by extracellularly administering PNA oligomers to living cells such that the PNA oligomers alter the expression of the polypeptide in a sequence specific manner and examining those cells for an activity that is influenced by the specific PNA oligomer. The PNA oligomers can be carrier-free and can be administered in vivo to an animal, such as a murine mammal. The PNA oligomers are capable of crossing biological barriers such as the plasma membrane of cells and the blood-brain barrier of a living organism. The PNA oligomers typically have sequence specificity for a nucleic acid sequence that encodes a polypeptide or regulates the expression of a polypeptide. The polypeptides can be expressed intracranially or extracranially in a living organism. Intracranially expressed polypeptides can include those polypeptides that participate in cell signaling. Cell signaling polypeptides include polypeptides that participate in opioid signaling. Opioid signaling polypeptides include opioid receptors, for example morphine and neurotensin receptors. Extracranially expressed polypeptides include polypeptides expressed outside the brain and cranium, for example in the gastrointestinal tract. Specific PNA oligomers can include oligomers having sequences such as set out in SEQ ID NO:s 1 and 2. The biological response engendered by the extracellular administration of PNA oligomers can be a modification, for example a reduction, of polypeptide expression. Biological responses also can be characterized by a physiological change in an animal.
Another aspect of the invention features a method of measuring the relative turnover rate of functional polypeptides having a defined activity by extracellularly administering PNA oligomers to living cells such that the PNA oligomers influence the defined activity, and determining the time after PNA oligomer administration that the defined activity is maximally influenced. In addition, the time from when the defined activity is influenced maximally to when the activity returns to normal can be determined.
Another aspect of the invention features an article of manufacture that combines packaging material and PNA oligomers. The packaging material includes a label or package insert indicating that the PNA oligomers can be extracellularly administered to living cells for the purpose of engendering a biological response in a sequence specific manner.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.